DESCRIPTION (from the applicant): Colonic epithelial cell differentiation is driven by reprogramming of stem and/or progenitor cells as they migrate upward along the crypt axis. This process is recapitulated in immortalized cell lines derived from human colon adenocarcinomas that proliferate in an immature state in culture yet can be induced by the short chain fatty acid (SCFA) butyrate (a product of dietary fiber fermentation in the human colon), or contact inhibition of growth to differentiate into absorptive (Caco-2), goblet (HT29 Cl16E), or secretory (HT29 Cl19A) colonic epithelial cells. Our group has provided key insights into mechanisms that drive this maturation by characterizing the gene expression profiles of these cell lineages as they differentiate. To identify genes for which altered expression is fundamental to the reprogramming of intestinal epithelial cells as they undergo maturation induced by physiological regulators such as butyrate, we focused on sequences that are consistently downregulated in expression during growth arrest and lineage specific differentiation of colonic epithelial cells in vivo. Suppression of one such gene, Mybl2, a molecule that has been suggested to regulate critical proliferation and differentiation decisions in stem cells and other cell types, by siRNA in proliferating colon epithelial cells induces expression of a subset of differentiation-specific genes. The aims of this proposal focus on determining whether Mybl2 modulates colon epithelial cell reprogramming, including that induced by the SCFA butyrate, by exerting parallel control over proliferation and differentiation pathways and elucidating how Mybl2 is itself regulated during colon cell maturation. Specific aims 1 and 2 will determine whether Mybl2 suppression can promote, or its overexpression can prevent, colon cell maturation through the coordinate regulation of proliferation and differentiation-specific genes. Specific aim #1 pursues our identification of a subset of genes, involved in promoting or preventing colon cell maturation, that is altered upon Mybl2 knockdown or overexpression, and specific aim #2 will reveal which gene promoters are bound and regulated by Mybl2 in maturing colon cells. Although transcriptional activation and repression account for many differentiation-specific gene expression changes, post-transcriptional regulation by microRNAs (miRNAs) provides another critical level of gene modulation during colon cell differentiation. We have found that the Mybl2 promoter is not significantly downregulated in differentiating colon epithelial cells and have therefore hypothesized that miRNAs may regulate its expression during maturation. We have utilized novel bioinformatic techniques to identify potential miRNA targets in the Mybl2 3'untranslated region (UTR). These potential targets: 1) are predicted by computer analysis to bind one or more known miRNAs and 2) form significant RNA secondary structure as indicated by a predicted free energy <0. These candidate miRNA target regions therefore have the potential to bind miRNA's that regulate fundamental mechanisms of differentiation in all three colon cell lineages. This target-centered approach will be used to identify specific miRNAs and their targets that regulate Mybl2 expression during colon cell differentiation. This approach, rather than a screening of expression of known miRNAs, will isolate and identify known and as well as unidentified miRNAs of biological relevance to colonic cell differentiation. Therefore, aim 3 will first determine whether predicted miRNA targets in the Mybl2 3'UTR are functional by assaying their ability to suppress luciferase expression when inserted into the 3'UTR of luciferase in differentiating colonic epithelial cells, as already indicated by preliminary data for one such predicted miRNA target. Functional targets will then be immobilized on streptavidin/agarose to physically capture miRNAs (known or novel) from small RNAs of differentiating colonic epithelial cells. The proposed studies will therefore reveal fundamental mechanisms utilized by several colon epithelial cell lineages to regulate the decision to proliferate or differentiate. PUBLIC HEALTH RELEVANCE: The secretory, absorptive, and protective functions of the adult colonic epithelium are essential for processing nutrients critical to the survival of cells and tissues throughout the body. Acquisition of these phenotypes relies on a complex process of differentiation that reprograms highly proliferative, multipotent stem cells into 3 specialized epithelial cell types (enterocytic, goblet, and enteroendocrine cells) as they migrate along the crypt axis towards the lumen and another (Paneth cells) that remains at the bottom of the crypt. How colon stem and/or precursor cells differentiate remains largely undefined. We do know that precursor cells undergo changes in several distinct categories of well-characterized genes. These changes can occur by a variety of ways, including by transcriptional activation and repression. Gene silencing by micro RNA's (miRNAs), likely plays a critical role as well because of the mounting evidence of their importance in development, differentiation, and disease. miRNAs silence gene expression by binding to target sequences within messenger RNAs (mRNAs) and inhibiting their translation into protein and/or initiating their degradation. Identifying these targets and characterizing how miRNAs can bind to them is critical to understand how miRNAs function in regulating gene expression that can promote normal processes such as differentiation or contribute to diseases such as cancer. Although the expression profiles of miRNAs in various cell lines and tissues are well characterized, very few functional interactions between miRNAs and their cognate mRNA targets in vivo are known. The overall goal of this proposal is to identify physiologically relevant targets of miRNAs that are involved in colonic epithelial cell differentiation and use them to isolate their cognate miRNAs from maturing colonic epithelial cells. These studies will not only elucidate previously unknown mechanisms of colonic epithelial cell maturation from stem cells into functional effectors but also have the potential to identify novel miRNAs. Furthermore, because a cell's decision to differentiate usually abrogates its tumorigenic potential, by identifying miRNAs that drive differentiation-specific gene expression we can discover cellular mechanisms involved in colorectal cancer prevention.